Bacillus subtilis probiotics and methods of use for improving immune function, hormonal status, and physical performance

ABSTRACT

Methods of using  Bacillus subtilis  probiotic supplementation to promote lower circulating TNF-α in resistance trained males are presented. Methods of administering  Bacillus subtilis  probiotic supplementation to an athletic population in conjunction with a sound nutrition and training regimen are presented herein. College athletes typically undergo periods of elevated stress both physically and mentally which may negatively affect recovery and adaptation. 12-weeks of probiotic supplementation resulted in attenuated circulating TNF-α concentrations in college athletes following offseason training.

This application claims the benefit of U.S. Provisional Application No.62/741,300 filed on Oct. 4, 2018, which is hereby incorporated byreference herein.

TECHNICAL FIELD

The present invention relates to methods of using Bacillus subtilis(DE111) probiotic supplementation to attenuate circulating markers ofinflammation and catabolism, e.g. TNF-α concentrations, in athletesfollowing offseason training and methods of administering Bacillussubtilis probiotic supplementation to an athletic population inconjunction with a sound nutrition and training regimen.

BACKGROUND

Athletes regularly engage in rigorous exercise training which leads toaccumulating amounts of physical stress. While daily moderate intensityphysical activity has been shown to have positive effects on the immunesystem (Petersen A M W and Pedersen B K. The anti-inflammatory effect ofexercise. Journal of applied physiology 98: 1154-1162, 2005), prolongedperiods of intense training and competition may lead to immunedysregulation (Gleeson M, McDonald W, Cripps A, Pyne D, Clancy R, andFricker P. The effect on immunity of long-term intensive training inelite swimmers. Clinical & Experimental Immunology 102: 210-216, 1995.;Mackinnon L T, Ginn E, and Seymour G J. Decreased salivaryimmunoglobulin A secretion rate after intense interval exercise in elitekayakers. European journal of applied physiology and occupationalphysiology 67: 180-184, 1993; Mackinnon L T and Jenkins D G. Decreasedsalivary immunoglobulins after intense interval exercise before andafter training. Medicine and science in sports and exercise 25: 678-683,1993). As a result of mucosal and systemic immune suppression, it iscommon for competitive athletes to become susceptible to infections,which may reduce the frequency and quality of physical training andathletic competition (Fahlman M M and Engels H-J. Mucosal IgA and URTIin American college football players: a year longitudinal study.Medicine and science in sports and exercise 37: 374-380, 2005). Inaddition to being vulnerable to infection, overly fatigued athletes arefound to have altered levels of pro and anti-inflammatory cytokines incirculation (Gepner Y, Hoffman J R, Shemesh E, Stout J R, Church D D,Varanoske A N, Zelicha H, Shelef I, Chen Y, and Frankel H. Combinedeffect of Bacillus coagulans GBI-30, 6086 and HMB supplementation onmuscle integrity and cytokine response during intense military training.Journal of Applied Physiology 123: 11-18, 2017; Hoffman J R, Gepner Y,Stout J R, Hoffman M W, Ben-Dov D, Funk S, Daimont I, Jajtner A R,Townsend J R, and Church D D. β-Hydroxy-β-methylbutyrate attenuatescytokine response during sustained military training. Nutrition research36: 553-563, 2016). For instance, elevated circulating TNF-α in elitemale rowers was significantly associated to depressed mood, sleepdisturbances, and physical stress (Main L C, Dawson B, Heel K, Grove JR, Landers G J, and Goodman C. Relationship between inflammatorycytokines and self-report measures of training overload. Research inSports Medicine 18: 127-139, 2010). Moreover, TNF-α acts to impairprotein synthesis in skeletal muscle by decreasing mRNA translationalefficiency (Lang C H, Frost R A, Nairn A C, MacLean D A, and Vary T C.TNF-α impairs heart and skeletal muscle protein synthesis by alteringtranslation initiation. American Journal of Physiology-Endocrinology andMetabolism 282: E336-E347, 2002). This combination of factors may limitan athlete's ability to properly recover from acute training bouts andmay ultimately impair training adaptations.

While athletes are often subjected to excessive levels of physicalstress as a byproduct of training demands, other stressors are oftenoverlooked. For instance, collegiate athletes regularly engage inperiods of high physical stress accompanied with prolonged travel,academic rigor, and other physiological stressors. College athletes whoare under a large amount of physical and academic stress have recentlybeen shown to be more susceptible to sustaining injury during thesetimes of increased strain (Mann J B, Bryant K R, Johnstone B, Ivey P A,and Sayers S P. Effect of physical and academic stress on illness andinjury in division 1 college football players. The Journal of Strength &Conditioning Research 30: 20-25, 2016). To counter this, biomarkermonitoring is gaining momentum in the athletic realm as a method todetect periods of excessive negative physiological stress (Lee E C,Fragala M S, Kavouras S A, Queen R M, Pryor J L, and Casa D J.Biomarkers in Sports and Exercise: Tracking Health, Performance, andRecovery in Athletes. Journal of strength and conditioning research 31:2920, 2017). Furthermore, it has been suggested that utilizing anassembly of diverse biomarkers may provide the most effective strategyin evaluating intricate balance of anabolic and catabolic processes inathletes (MacKinnon LT. Overtraining effects on immunity and performancein athletes. Immunology & Cell Biology 78: 502-509, 2000; Smith L L.Cytokine hypothesis of overtraining: a physiological adaptation toexcessive stress? Medicine & Science in Sports & Exercise 32: 317,2000).

To attenuate the increasing levels of physiological strain associatedwith training, athletes often implement nutritional strategies tosupport immune health. Probiotic supplementation, for instance, is astrategy which is receiving considerable attention as a countermeasurefor training-induced stressors (Pyne D B, West N P, Cox A J, and CrippsA W. Probiotics supplementation for athletes—clinical and physiologicaleffects. European journal of sport science 15: 63-72, 2015). Probioticsare live organisms that when consumed, impose a wide array of beneficialphysiological effects on humans, most notably promoting improved gutmicrobiota (Borchers A T, Selmi C, Meyers F J, Keen C L, and Gershwin ME. Probiotics and immunity. Journal of gastroenterology 44: 26-46,2009). These microorganisms have been shown to exert immunomodulatoryeffects (Lescheid D W. Probiotics as regulators of inflammation: Areview. Functional foods in health and disease 4: 299-311, 2014) bydecreasing pro-inflammatory cytokines in circulation (Lamprecht M,Bogner S, Schippinger G, Steinbauer K, Fankhauser F, Hallstroem S,Schuetz B, and Greilberger J F. Probiotic supplementation affectsmarkers of intestinal barrier, oxidation, and inflammation in trainedmen; a randomized, double-blinded, placebo-controlled trial. Journal ofthe International Society of Sports Nutrition 9: 45, 2012) andsupporting mucosal defense (Gleeson M, Bishop N C, Oliveira M, andTauler P. Daily probiotic's (Lactobacillus casei Shirota) reduction ofinfection incidence in athletes. International journal of sportnutrition and exercise metabolism 21: 55-64, 2011; Michalickova D, MinicR, Dikic N, Andjelkovic M, Kostic-Vucicevic M, Stojmenovic T, Nikolic I,and Djordjevic B. Lactobacillus helveticus Lafti L10 supplementationreduces respiratory infection duration in a cohort of elite athletes: arandomized, double-blind, placebo-controlled trial. Applied Physiology,Nutrition, and Metabolism 41: 782-789, 2016). As probiotics havepreviously been shown to modulate pro- and anti-inflammatory cytokinesin the body, it has been suggested that probiotics may support anathlete's general immune health (Pyne, et al., 2015). Additionally,intense physical training may cause damage to an athlete's gut barrier,resulting in endotoxin translocation, oxidative stress, and a low-gradepro-inflammatory cytokine response (Lamprecht, et al., 2012; MartarelliD, Verdenelli M C, Scuri S, Cocchioni M, Silvi S, Cecchini C, and PompeiP. Effect of a probiotic intake on oxidant and antioxidant parameters inplasma of athletes during intense exercise training. Currentmicrobiology 62: 1689-1696, 2011; Pugh J N, Impey S G, Doran D A,Fleming S C, Morton J P, and Close G L. Acute high-intensity intervalrunning increases markers of gastrointestinal damage and permeabilitybut not gastrointestinal symptoms. Applied Physiology, Nutrition, andMetabolism 42: 941-947, 2017; Van Wijck K, Lenaerts K, Van Loon L J,Peters W H, Buurman W A, and Dejong C H. Exercise-induced splanchnichypoperfusion results in gut dysfunction in healthy men. PloS one 6:e22366, 2011). In athletes, probiotics have been reported to reduce thenumber, duration, and severity of infections (Gleeson, et al., 2011; CoxA J, Pyne D B, Saunders P U, and Fricker P A. Oral administration of theprobiotic Lactobacillus fermentum VRI-003 and mucosal immunity inendurance athletes. British Journal of Sports Medicine 44: 222-226,2010; West N P, Pyne D B, Cripps A W, Hopkins W G, Eskesen D C, JairathA, Christophersen C T, Conlon M A, and Fricker P A. Lactobacillusfermentum (PCC®) supplementation and gastrointestinal andrespiratory-tract illness symptoms: a randomised control trial inathletes. Nutrition Journal 10: 30, 2011). Thus by improving resistanceto infection, attenuating low-grade inflammation, and improving nutrientabsorption, probiotic supplementation may be a practical strategy tosupport athlete health and adaptation (Pyne, et al., 2015; Coqueiro A Y,de Oliveira Garcia A B, Rogero M M, and Tirapegui J. Probioticsupplementation in sports and physical exercise: Does it present anyergogenic effect? Nutrition and health 23: 239-249, 2017).

While probiotic supplementation appears to have a generally positiveeffect on athlete immune function, its efficacy on improving exerciseperformance is less clear. In endurance athletes, a multi-strainprobiotic significantly improved time until fatigue in males running at80% of their ventilatory threshold (Shing C M, Peake J M, Lim C L,Briskey D, Walsh N P, Fortes M B, Ahuja K D, and Vitetta L. Effects ofprobiotics supplementation on gastrointestinal permeability,inflammation and exercise performance in the heat. European journal ofapplied physiology 114: 93-103, 2014) whereas others have reported noeffect of probiotics on performance (Cox, et al., 2010; Michalickova, etal., 2016; West, et al., 2011). Regarding resistance exercise, Jager, etal., found that co-ingestion of protein with a Bacillus strain probioticattenuated range of motion decrements in recovery following an intensebout of resistance exercise possibly by improving nutrient absorption(Jager R, Purpura M, Stone J D, Turner S M, Anzalone A J, Eimerbrink MJ, Pane M, Amoruso A, Rowlands D S, and Oliver J M. ProbioticStreptococcus thermophilus FP4 and Bifidobacterium breve BR03supplementation attenuates performance and range-of-motion decrementsfollowing muscle damaging exercise. Nutrients 8: 642, 2016; and alsoKeller D, Van Dinter R, Cash H, Farmer S, and Venema K. Bacilluscoagulans GBI-30, 6086 increases plant protein digestion in a dynamic,computer-controlled in vitro model of the small intestine (TIM-1).Beneficial microbes 8: 491-496, 2017). Furthermore, 10-weeks of Bacillussubtilis supplementation in conjunction with adequate post-workoutnutrition was shown to improve body composition in female collegiateathletes (Toohey, J C, Townsend J R, Johnson S B, Toy A M, Vantrease WC, Bender D, Crimi C C, Stowers K L, Ruiz M D, VanDusseldorp T A, FeitoY, Mangine G T, “The effects of probiotic (Bacillus subtilis)supplementation during offseason resistance training in female DivisionI athletes,” The Journal of Strength and Conditioning Research, (2018,in press).

Despite recent interest, the effects of probiotic supplementation ontraining outcomes in resistance-trained individuals and on theirperformance is still unclear (Georges J, Lowery R P, Yaman G, Kerio C,Ormes J, McCleary S A, Sharp M, Shields K, Rauch J, and Silva J. Theeffects of probiotic supplementation on lean body mass, strength, andpower, and health indicators in resistance trained males: a pilot study.Journal of the International Society of Sports Nutrition 11, 2014;Ibrahim N S, Muhamad A S, Ooi F K, Meor-Osman J, and Chen C K, “Theeffects of combined probiotic ingestion and circuit training on muscularstrength and power and cytokine responses in young males,” AppliedPhysiology, Nutrition, and Metabolism (2018) 43: 180-186).

SUMMARY OF THE INVENTION

In an embodiment, the present invention examines the effect of dailyprobiotic supplementation on strength, performance, body composition andbiochemical markers in Division I male college athletes. The testresults indicate that probiotic supplementation may provide additionalbenefits on strength, performance, and body composition followingoffseason training compared to placebo (“PL”). Furthermore, probioticsupplementation promotes lower circulating TNF-α in resistance trainedmales. These findings provide a method of administering probioticsupplementation in an athletic population in conjunction with a soundnutrition and training regimen.

In another embodiment, a method is described for improving physicalperformance in a human, comprising the steps of: (a) administeringorally to the human a composition comprising Bacillus subtilis in a doseof from about 1·10⁸ CFU per day to about 1·10¹¹ CFU per day; and (b)measuring TNF-α level in blood plasma of the human. The administeringstep can be performed for about 10-12 weeks.

In another embodiment, a method of reducing TNF-α in human serum isdescribed, comprising the steps of: (a) administering orally to thehuman a composition comprising Bacillus subtilis in a dose of from about1·10⁸ CFU per day to about 1·10″ CFU per day for about 12 weeks; and (b)submitting the human to a resistance training program throughout the 12weeks. The resistance program may include workouts 2-3 times per week.

DETAILED DESCRIPTION

In its principal embodiment, a Bacillus subtilis (B. subtilis)containing composition is used for probiotic supplementation in a humansubject. One useful Bacillus subtilis-containing composition is DE111®available from Deerland Enzymes, Inc. (Kennesaw, Ga.). The probioticsupplement can include 5 billion CFU Bacillis subtilis.

As described herein, DE111® may be used in an effective total daily dosefrom about 1×10⁸ CFU to about 1×10¹¹ CFU. One preferred daily dose rangeis from about 1×10⁹ CFU to about 1×10¹⁰ CFU.

The Bacillus subtilis DE111 strain has certain properties, which,surprisingly, have been found to make the strain well-suited for use asa probiotic. Spores of Bacillus subtilis are viable under a widetemperature and pH range. Without being bound by any particular theory,it is thought that the ability of Bacillus subtilis DE111 to form sporesthat protect the microbes from harsh conditions until they enter anenvironment ripe for germination, such as the GI tract, makes Bacillusparticularly well-suited for use as a probiotic.

In one aspect of the invention, compositions administered to patients inneed thereof according to the methods of the present disclosure comprisemutants of Bacillus subtilis DE111 having all the identifyingcharacteristics of Bacillus subtilis DE111. Such mutants may have DNAsequence identity to Bacillus subtilis DE111 of at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99%. In some embodiments, mutants arespontaneous mutants. The teen spontaneous mutant refers to mutants thatarise from Bacillus subtilis DE111 without the intentional use ofmutagens. Such spontaneous mutants may be obtained by classical methods,such as growing the Bacillus subtilis DE111 strain in the presence of acertain antibiotic to which the parent is susceptible, and testing anyresistant mutants for improved biological activity or, in thisapplication, ability to improve the body composition of an individual.Other methods for identifying spontaneous mutants will be known to thoseof ordinary skill in the art.

All references in this application to Bacillus subtilis DE111 or itsmutants refer to bacteria that have been isolated from nature and aregrown by humans, for example, in the laboratory or under industrialconditions.

Bacillus subtilis DE111 cells may be present in the compositionsadministered to patients in need thereof according to the methods of thepresent disclosure as spores (which are dormant), as vegetative cells(which are growing), as transition state cells (which are transitioningfrom growth phase to sporulation phase) or as a combination of all ofthese types of cells. In some embodiments, the composition comprisesmainly spores. In other embodiments, the composition comprises sporesand metabolites produced by the cells during fermentation before theysporulate, as described below.

Compositions administered to patients in need thereof according to themethods of the present disclosure can be obtained by culturing Bacillussubtilis DE111 or its mutants according to methods well known in theart. Conventional large-scale microbial culture processes includesubmerged fermentation, solid state fermentation, or liquid surfaceculture. Towards the end of fermentation, as nutrients are depleted,Bacillus subtilis DE111 cells begin the transition from growth phase tosporulation phase, such that the final product of fermentation islargely spores, metabolites, and residual fermentation medium.Sporulation is part of the natural life cycle of Bacillus subtilis DE111and is generally initiated by the cell in response to nutrientlimitation. Fermentation is configured to obtain high levels of colonyforming units of Bacillus subtilis DE111 and to promote sporulation. Thebacterial cells, spores, and metabolites in culture media resulting fromfermentation may be used directly or concentrated by conventionalindustrial methods, such as centrifugation, tangential-flow filtration,depth filtration, and evaporation. In some embodiments, the concentratedfermentation broth is washed, for example, via a diafiltration process,to remove residual fermentation broth and metabolites.

The fermentation broth or broth concentrate can be dried with or withoutthe addition of carriers using conventional drying processes or methodssuch as spray drying, freeze drying, tray drying, fluidized-bed drying,drum drying, or evaporation. The resulting dry products may be furtherprocessed, such as by milling or granulation, to achieve a specificparticle size or physical format. Carriers, described below, may also beadded post-drying.

In embodiments in which compositions formulated separately from food ordrink are administered to patients in need thereof according to themethods of the present disclosure, the concentration on a weight byweight basis (w/w) of (i) Bacillus subtilis DE111 or its mutants, (ii)metabolites of Bacillus subtilis DE111 or its mutants, or (iii)combinations of cells and metabolites in the formulated composition maybe about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%. In some embodiments of compositionsadministered to patients in need thereof according to the methods of thepresent disclosure, where the concentrated formulation broth has beenwashed and dried without heat, such as via freeze drying, theconcentration of Bacillus subtilis DE111 or its mutants in the finalcomposition may be from about 90% to about 100%.

In certain embodiments, compositions administered to individuals in needthereof according to the methods of the present disclosure areadministered to improve strength, performance and body composition. Aneffective amount of a composition administered to an individual in needthereof according to the methods of the present disclosure is an amounteffective to improve body composition in comparison to an individual whohas not been administered the composition but otherwise has beenadministered the same diet as has an individual administered thecomposition according to methods of the present disclosure. In otherembodiments, an effective amount of a composition administered to anindividual in need thereof according to the methods of the presentdisclosure is an amount effective to reduce body fat percentage incomparison to an individual who has not been administered thecomposition but otherwise has been administered the same diet as has anindividual administered the composition according to the methods of thepresent disclosure.

Thus, in line with the above, embodiments of the present disclosure aredirected to methods of improving body composition, and/or reducing bodyfat percentage, by administering to an individual in need thereof acomposition comprising Bacillus subtilis DE111, a mutant of Bacillussubtilis DE111, metabolites of Bacillus subtilis DE111 or its mutants,or combinations of Bacillus subtilis DE111 or a mutant and metabolitesof Bacillus subtilis DE111 or its mutants.

Without wishing to be bound by any particular theory, it is thought thatincreases to beneficial bacteria may be caused by stimulating growth ofsuch bacteria or simply by selectively decreasing pathogenic bacteria,thereby giving the beneficial bacteria more space to grow and to attachto the gut wall and/or more efficient access to nutrients and growthfactors. In addition, or alternatively, beneficial bacteria may modifythe virulence factors of pathogenic bacteria, thus decreasing thevirulence of the pathogenic bacteria. Harmful, disease-causing bacteriathat may be decreased by the methods of the present disclosure includeClostridia spp. (such as perfringens and dificille), Listeria spp. (suchas Moncytogenes, seeligeri, and welshimeri), Salmonella spp. (such asenterica, arizonae, typhirium, enteridis, and bonglori), E. coli,Enterococus spp. (such as faecalis and faecium), Camphylobacter,Aeromonas spp., Staphylococcus aureus, Shigella dysenteria, and Vibriospp. In some embodiments, harmful, disease-causing microorganisms may bereduced by about 0.5 log, about 1 log, about 2 log, about 3 log, about 4log, or about 5 log.

In another aspect, compositions administered according to methods of thepresent disclosure comprising Bacillus subtilis DE111, its mutants,and/or metabolites of Bacillus subtilis DE111 and/or its mutants mayfurther include or be administered with other probiotics, such as otherbacterial spore formers. Examples of probiotics are provided in H. A.Hong, et al., The use of bacterial spore formers as probiotics, 29 FEMSMICROBIOLOGY REVS. 813 (2005), incorporated by reference herein in itsentirety.

In yet another aspect, compositions administered according to methods ofthe present disclosure may include or be administered with (either atthe same time or at different times) anti-diarrheal agents, anti-gasagents, dietary fibers, antibiotics, such as methotrexate,anti-inflammatory drugs, amino acids, electrolytes, vitamins, andminerals.

In embodiments in which the compositions administered according tomethods of the present disclosure comprise Bacillus subtilis DE111 orits mutants, the bacteria should be administered in an amount that iseffective to improve body composition and/or reduce body fat percentage.In embodiments in which the compositions are being administered toimprove body composition and/or reduce body fat percentage, thecompositions should be administered at effective total daily doses offrom about 1·10³ CFU Bacillus subtilis DE111 to about 1·10¹⁵ CFUBacillus subtilis DE111. In other embodiments in which the compositionsare being administered to improve body composition and/or reduce bodyfat percentage, the compositions should be administered at effectivetotal daily doses of from about 1·10⁴ CFU Bacillus subtilis DE111 toabout 1·10¹⁴ CFU Bacillus subtilis DE111. In yet other embodiments inwhich the compositions are being administered to improve bodycomposition and/or reduce body fat percentage, the compositions shouldbe administered at effective total daily doses of from about 1·10⁵ CFUBacillus subtilis DE111 to about 1·10¹³ CFU Bacillus subtilis DE111. Inyet other embodiments in which the compositions are being administeredto improve body composition and/or reduce body fat percentage, thecompositions should be administered at effective total daily doses offrom about 1·10⁶ CFU Bacillus subtilis DE111 to about 1·10¹² CFUBacillus subtilis DE111. In yet other embodiments in which thecompositions are being administered to improve body composition and/orreduce body fat percentage, the compositions should be administered ateffective total daily doses of from about 1·10⁸ CFU Bacillus subtilisDE111 to about 1·10¹¹ CFU Bacillus subtilis DE11. In yet otherembodiments, a preferred effective total daily dose range is from about1·10⁹ CFU Bacillus subtilis DE111 to about 1·10¹⁰ CFU Bacillus subtilisDE111. In yet another embodiment, Bacillus subtilis DE111 can beprovided in a daily dose of about 5·10⁹ CFU for several weeks, up to atotal of about 10-12 weeks.

In an embodiment, administration of a Bacillus subtilis DE111 dose atabout 5 billion CFU per day statistically improved body compositionand/or statistically reduced body fat percentage of an individual. Incontrast, the testing group administered placebo composition did notgenerate similar improvements.

In certain embodiments, the compositions administered according to themethods of the present disclosure may also include one or moreexcipients, most preferably one or more nutraceutical or pharmaceuticalexcipients. Compositions containing one or more excipients andincorporating one or more probiotics can be prepared by procedures knownin the art. Optionally, compositions can include one or more adjuvants,excipients, carriers, buffers, diluents, and/or other customarypharmaceutical auxiliaries. For example, probiotics can be formulatedinto tablets, capsules, powders, suspensions, solutions for oraladministration, solutions for parenteral administration includingintravenous, intradermal, intramuscular, and subcutaneousadministration, and solutions for application onto patches fortransdermal application with common and conventional barriers, binders,diluents, and excipients.

In certain embodiments, nutraceutical compositions administeredaccording to the methods of the present disclosure may be administeredin combination with a pharmaceutically acceptable carrier. In certainembodiments, the active ingredients in such formulations may comprisefrom about 1% by weight to about 99% by weight. In other embodiments,the active ingredients in such formulations may comprise from about 0.1%by weight to about 99.9% by weight. “Pharmaceutically acceptablecarrier” means any carrier, diluent, or excipient that is compatiblewith the other ingredients of the formulation and not deleterious to theuser. Useful excipients include, but are not limited to,microcrystalline cellulose, magnesium stearate, calcium stearate, anyacceptable sugar (e.g., mannitol, xylitol), and the like, and forcosmetic use, an oil-base is preferred.

In another embodiment, the dose of DE111 can be provided in a daily doseof about 1×10⁹ CFU for several weeks, up to a total of about 12 weeks.

One embodiment of the present invention relates to the effects of12-week of daily probiotic supplementation on the immune and hormonalprofile in college athletes during a period of increased academic andphysical stress. Another embodiment of the present invention relates tothe effects of daily probiotic supplementation on physical andperformance adaptations in Division I collegiate baseball playersfollowing 12-weeks of offseason training.

TNF-α is a potent pro-inflammatory cytokine which is designed to servean essential role in skeletal muscle remodeling. However, pronouncedlevels of TNF-α have been linked suppressed protein synthesis,disordered sleep, and impaired muscular performance. Probioticsupplementation having different bacterial strains from those disclosedherein reduced circulating TNF-α concentrations in endurance trained men(Lamprecht, et al., 2012) while West, et al., (2011) found thatprobiotic supplementation likely decreased the magnitude of TNF-αconcentrations following acute VO₂max testing.

IL-10 is an anti-inflammatory cytokine, which is generally elevated postresistance exercise as a means to suppress inflammation and begin theadaptation process (Hirose L, Nosaka K, Newton M, Laveder A, Kano M,Peake J, and Suzuki K. Changes in inflammatory mediators followingeccentric exercise of the elbow flexors. Exerc Immunol Rev 10: 20, 2004;Peake J, Nosaka K K, and Suzuki K. Characterization of inflammatoryresponses to eccentric exercise in humans. 2005). Ibrahim, et al.,(2018) using different bacterial strains from the disclosed inventionfound a significant increase in IL-10 concentrations following 12 weeksof circuit training alone and probiotic supplementation alone while thecombination of circuit training and probiotics trended towards asignificant elevation post intervention.

Immunoglobulins are a heterogeneous group of antimicrobial proteinswhich appear as the immune system's first line of defense in theresponse to an antigen (Trochimiak T and Hübner-Woźniak E. Effect ofexercise on the level of immunoglobulin A in saliva. Biology of sport29: 255, 2012). IgA is the principal immunoglobulin involved in hostdefense and has been shown to be suppressed following intense acute(Mackinnon L T, Chick T W, Van As A, and Tomasi T B. Decreased secretoryimmunoglobulins following intense endurance exercise. Research in SportsMedicine: An International Journal 1: 209-218, 1989; Mackinnon L T, GinnE, and Seymour G J. Decreased salivary immunoglobulin A secretion rateafter intense interval exercise in elite kayakers. European journal ofapplied physiology and occupational physiology 67: 180-184, 1993;Steerenberg P A, Asperen I A, Amerongen A N, Biewenga J, Mol D, andMedema G. Salivary levels of immunoglobulin A in triathletes. Europeanjournal of oral sciences 105: 305-309, 1997) and chronic training(Gleeson, et al., 2011; Mackinnon L T and Jenkins D G. Decreasedsalivary immunoglobulins after intense interval exercise before andafter training. Medicine and science in sports and exercise 25: 678-683,1993). Contrary to reports in endurance athletes (Gleeson, et al., 2011)and military cadets (Tiollier E, Chennaoui M, Gomez-Merino D, Drogou C,Filaire E, and Guezennec C Y. Effect of a probiotics supplementation onrespiratory infections and immune and hormonal parameters during intensemilitary training. Military medicine 172: 1006-1011, 2007), a number ofstudies in endurance athletes found no differences in SIgA or SIgMbetween groups following probiotic interventions ranging from 4-12 weeks(Clancy R, Gleeson M, Cox A, Callister R, Dorrington M, D′este C, PangG, Pyne D, Fricker P, and Henriksson A. Reversal in fatigued athletes ofa defect in interferon γ secretion after administration of Lactobacillusacidophilus. British journal of sports medicine 40: 351-354, 2006; Cox,et al., 2010; West, et al., 2011). Additionally, another study found nodifferences in SIgA protein concentration or secretion rate in 24 maleand 6 female professional athletes of various sports (Michalickova, etal., 2016).

Testosterone and cortisol represent a hormonal parameters which providea snapshot of the current anabolic status of an athlete (Lee, et al.,2017). Traditionally, these two endocrine biomarkers are utilized inmale athletes to identify and prevent overtraining (Hayes L D, Grace FM, Baker J S, and Sculthorpe N. Exercise-induced responses in salivarytestosterone, cortisol, and their ratios in men: a meta-analysis. SportsMedicine 45: 713-726, 2015). Previous work found no effect of probioticson cortisol concentrations during a period of intense military training(Tiollier, et al., 2007). However, one study in a non-athleticpopulation reported lower cortisol responses in participants whoreceived a prebiotic (soluble fiber compounds which enhance the growthof gut microbiota) supplement daily for 3 weeks (Schmidt K, Cowen P J,Harmer C J, Tzortzis G, Errington S, and Burnet P W. Prebiotic intakereduces the waking cortisol response and alters emotional bias inhealthy volunteers. Psychopharmacology 232: 1793-1801, 2015).

Zonulin is a protein, which plays a central role in modulatingintercellular tight junctions in the intestinal endothelium. Of late,this protein has been proposed as a novel circulating marker ofintestinal permeability. Previous work found that 14 weeks of probioticsupplementation resulted in significantly decreased levels of fecalzonulin, indicating an improvement in intestinal barrier integrity(Lamprecht, et al., 2012). Some investigations have observed compromisedgut permeability in response to an acute exercise stress in trainedparticipants following endurance and interval training (Mach N andFuster-Botella D. Endurance exercise and gut microbiota: A review.Journal of sport and health science 6: 179-197, 2017; Pugh, et al.,2017). Clarke et al., (Clarke S F, Murphy E F, O'sullivan O, Lucey A J,Humphreys M, Hogan A, Hayes P, O'reilly M, Jeffery I B, and Wood-MartinR. Exercise and associated dietary extremes impact on gut microbialdiversity. Gut: gutjnl-2013-306541, 2014) found that trained athletespossess a healthier, more diverse gut microbiota.

It has been proposed probiotic supplementation may improvegastrointestinal function resulting in increased absorption of dietaryprotein (Keller D, Van Dinter R, Cash H, Farmer S, and Venema K.Bacillus coagulans GBI-30, 6086 increases plant protein digestion in adynamic, computer-controlled in vitro model of the small intestine(TIM-1). Beneficial microbes 8: 491-496, 2017), which may contribute toenhanced adaptations over the course of a training intervention. In amouse model, 6-weeks of Lactobacillus plantarum produced augmentedstrength, muscle mass, and type I muscle fiber number while improvingendurance swimming performance (Chen Y-M, Wei L, Chiu Y-S, Hsu Y-J, TsaiT-Y, Wang M-F, and Huang C-C. Lactobacillus plantarum TWK10supplementation improves exercise performance and increases muscle massin mice. Nutrients 8: 205, 2016).

To date, only two studies have investigated the effect of probioticadministration on resistance training adaptations. The firstinvestigation (Ibrahim, et al., 2018) found no ergogenic benefit of aprobiotic supplement on muscular strength and power following 12 weeksof circuit-resistance training which is in concert with previous work inendurance athletes reporting no effect of probiotics on performance(Cox, et al., 2010; Gill S K, Teixeira A M, Rosado F, Cox M, and Costa RJ S. High-dose probiotic supplementation containing Lactobacillus caseifor 7 days does not enhance salivary antimicrobial protein responses toexertional heat stress compared with placebo. International journal ofsport nutrition and exercise metabolism 26: 150-160, 2016; Michalickova,et al., 2016; West, et al., 2011). The second study found nopreferential benefit of daily Bacillus subtilis (5 billion CFU)supplementation on measures of physical performance following 10-weeksof offseason training in female Division I volleyball and soccerathletes (Toohey, et al., 2018). However, Toohey, et al. (2018) didobserve significant improvements in body compositions which mimickedthose seen in non-athletic populations.

The methods described above may be further understood in connection withthe following Examples. In addition, the following non-limiting examplesare provided to illustrate the invention. However, the person skilled inthe art will appreciate that it may be necessary to vary the proceduresfor any given embodiment of the invention, e.g., vary the order orsteps.

Example 1. Methods and Study Protocols

Methods

Twenty-five Division I male baseball athletes (20.1±1.5 y, 85.5±10.5 kg,184.7±6.3 cm) participated in this double blind, placebo-controlled,randomized study. Participants were randomly assigned to a probiotic(PRO; n=13) or placebo (PL; n=12) group. Following an explanation of allprocedures, risks, and benefits, each participant provided their writteninformed consent prior to participation in this study. The researchprotocol was approved by the Institutional Review Board of the LipscombUniversity prior to participant enrollment. Exclusion criteria includedthe use of probiotic supplementation, ergogenic aids, or suffering fromany medical, muscular, or metabolic contraindications.

Study Protocol

Participants reported to the Human Performance Lab (HPL) on two separateoccasions at the beginning and end of the 12-week training interventionfollowing a 10-hour overnight fast. Additionally, athletes wereinstructed to report to the lab hydrated while abstaining from caffeine,alcohol, and vigorous exercise for at least 24 h prior to bothlaboratory testing sessions. During these visits the participants weretested for body composition, muscle thickness, and provided biologicalsamples. Furthermore, athletes reported to their strength andconditioning coordinator on two separate occasions pre and posttraining, to measure one repetition maximum (“1RM”) for squat anddeadlift along with testing pro-agility, 10-yd sprint, and standing longjump. Pre-training all 1RM sessions began at the beginning of the fallsemester the first week of classes. Post-training, 1RM and performancetesting occurred the week prior to final examinations. Since the aim wasto investigate biomarkers of fatigue and immune function during astressful period, we chose to conduct our post-training biochemicalsample collection during final examination week (Mann, et al., 2016).Additionally, as winter months have been shown to produce additionalchallenges to the immune system (West, et al., 2011), our post-testingbiochemical sampling occurred in a winter month as well (December).

Supplementation Protocol

Both the PRO and PL groups completed daily supplementation for 12 weeks.The PRO supplement consisted of 1 billion colony forming units (CFU)Bacillis subtilis, (DE111®, Deerland Enzymes, Kennesaw, Ga., USA). Ontraining days, supplementation occurred immediately post-workout with aprotein and carbohydrate recovery drink (27 g protein, 36 gcarbohydrates, 2 g fat) in the presence of a study investigator. Onweekend or non-training days, athletes were provided their respectivesupplements in individual bags and were required to consume theirsupplement with a normal meal and return the used supplement bags toestablish compliance.

Nutritional Analysis

During the training and supplement intervention participants were askedto complete a three-day food log (two weekdays, one weekend day) onweeks one, nine, and 12. Dietary recalls were used to provide anestimate of total kilocalorie intake (kcal) and macronutrientdistributions (carbohydrate, protein, and fat) of the athlete's typicalweekly diet. All dietary analysis was completed using the MyFitnessPalapplication (Under Armour Inc., Baltimore, Md., USA), which contains alarge, detailed US-branded food database.

Statistical Analysis

Prior to hypothesis testing, the Shapiro-Wilk test was used to evaluatethe assumption of normality for dependent variables. Non-normallydistributed data were transformed using the natural log. To identifydifferences between the experimental conditions on changes in musclesize and strength, an ANCOVA was performed on all measures collected atPOST. Associated values collected at PRE were used as the covariate toeliminate the possible influence of initial score variances on theoutcomes. Following any significant F-ratio, a paired-samples t-test wasused to determine if significant difference existed between measurescollected prior to and immediately following 12 weeks of training. Groupdifferences were further assessed via effect sizes (rep; partial etasquared). Effect sizes were interpreted as small (0.01-0.059), medium(0.06-0.139), or large (>0.14) as previously recommended (Green S,Salkind N, and Akey T. Methods for controlling type I error acrossmultiple hypothesis tests. Using SPSS for Windows: Analysing andUnderstanding Data: 395-396, 2000). An alpha level was set at p≤0.05,and all analyses were performed using SPSS version 24.0 (SPSS, Inc.,Chicago, Ill.).

Offseason Training

All athletes completed the same triphasic undulating periodizedresistance training program for 12 weeks (2-3 days·week⁻¹) (Table 1).Triphasic training is a common periodized resistance training programdesigned to allow an athlete to eccentrically and isometrically absorbenergy before applying it in a dynamic movement (Dietz C and Peterson B.Triphasic training: A systematic approach to elite speed and explosivestrength performance. Bye Dietz Sport Enterprise, 2012). This programconsists of three mesocyles (3-4 weeks) in which athletes emphasize aparticular phase of movement (eccentric, isometric, concentric) whileperforming their core lifts. In addition to strength training, theathletes participated in team conditioning, agility, jumping, and sprintwork (2-3 sessions·week⁻¹). These workouts consisted of approximately30-40 minutes of sport-specific skill development andconditioning-related work. All training sessions were performed underthe supervision of a certified strength and conditioning specialist aswell as a certified athletic trainer.

TABLE 1 12-week Offseason Resistance Training Program Phase 1-EccentricWeeks 1-4 Day 1 Sets × Reps Day 2 Sets × Reps Day 3 Sets × Reps Squat 4× 8-5 Dead Lift 4 × 8-5 Hang Clean 4 × 8-5 Box jump w/;03-;05 ECC SingleHops 4 × :08 seconds Single Leg Box Jumps 4 × 5 4 × 4 Single Leg Box 4 ×5 Inverted Row 4 × 10 Mobility 3 × 10 Squats Single Arm Dumbbell 4 × 8-6Bench Press 4 × 8-5 Scap Angels 3 × 10 Bench 3 Point Row w/;03-;05 ECCDumbbell Incline 4 × 8-4 Exercise Ball Core 4 × 6 4 × 8 Press Sled Push4 × 1 GHD Falls w/;03-;05 ECC Banded Swimmers 4 × 10 Banded Hip FlexerPull 4 × 10 3 × 8 Row Banded Face Pull 4 × 10 6 Pack Scaps YTA 3 × 6:03ECC Circuit 1 50:10 × 3 Circuit 2 Circuit 1 Circuit 2 Circuit 1 Circuit2 Int/Ext Shoulder Rotation Split Squat Airex Floor Touches KeiserResisted Band Pull-Aparts Box Step-ups Lunge Plank TGU Banded Hip LiftsBanded × Walks Keiser SL Twist Ab Wheel HK Chops Pullup Shoulder RaisesSide Plank Row Kettle Bell Lunge Med Ball Slams Phase 2-Isometric Weeks5-8 Day 1 Sets × Reps Day 2 Sets × Reps Hang Clean 4 × 6-4 Dead Lifts 4× 6-4 Mobility 3 × 5 SL Hexagon Hops 4 × :08 4 × 6-4 W/Y Negatives 3 × 8Squat w/;03-;05 ISO SL Pistol Squat 4 × 5 Lateral Box Jump 4 × 4 BenchPress 4 × 6-4 w/:03 ISO DB Incline Bench Press 4 × 6-4 Battle Rope 3 ×:30 Bear Row w/;03 ISO Variations 4 × 8-6 Black Burns 3 × 5 Sled Pushw/;03 ISO SL RDL Reaches 3 × 8 3 × 1 TRX Archor Row 3 × 8 Lateral Lunge3 × 8 Landmine Rotation 3 × 8 Farmers Carry 3 × 3 and Press Pull-ups 2 ×8, 1 × 6 Med Ball Fielding 3 × 10 Standing Keiser Twists w/;03 ISO Drill3 × 10 Excercise Ball Knee 3 × 10 Drives Phase 3-Concentric Weeks 9-12Day 1 Sets × Reps Day 2 Sets × Reps Day 3 Sets × Reps Squat 4 × 4-2 DeadLift 4 × 4-2 Hang Clean 4 × 4-2 Box Jump 4 × 4 lateral Bound 4 × 6 DeadBugs 4 × 5 Mobility 3 × 5 Inverted Row 3 × 8 Cross-Over ATYT 3 × 15Incline Bench 4 × 4-3 Bench Press 4 × 4-3 Mobility 3 × 10 3 Point Row 4× 5-3 Med Ball Chest Pass 4 × 5 Single Arm Bench 4 × 4-3 Hip Lift 4 × 6BlackBurns 4 × 5 6 Pack Scaps 4 × 6 Battle Rope Variations 3 × :30Single Leg Squat 4 × 5 Lateral Sled Pull 3 × 1 Inline Board Lunge 3 × 5Side Plank Row 3 × 8 Keiser Single Arm 3 × 8 Pull-up 3 × 8 BandPull-Aparts 3 × 10 Single Leg Row Keiser Low Row 3 × 8 Val Slide Lateral3 × 8 Med Ball Slams 3 × 10 Supine Bridge w/ Cross 3 × 10 Lunge TowelPull-ups 3 × 8 Body Med Ball Throw Landmine Touches 3 × 10 Vertimax PullOver 3 × 10 Prone Hip Openers 3 × 10

Results

No significant differences were observed between groups for compliance,with all athletes achieving≥92% with an average compliance of 98.8%across groups. No significant differences in average daily caloricintake were observed between PRO (2404±494.3 kcals) and PL (2369±616.3kcals) groups. In addition, no significant differences were seen betweengroups in carbohydrate (PRO: 262.2±52.3 g vs. PL: 251.4±62.6 g), protein(PRO: 122.3±33.3 g vs. PL: 128.0±40.1 g) and fat (PRO: 91.3±28.7 g vs.PL: 86.5±24.1 g) intakes. Furthermore, both PRO and PL supplements werewell tolerated, and no adverse side effects were reported.

Example 2. Body Composition, Muscle Density, Strength and PhysicalPerformance

Body Composition

Air Displacement Plethysmography

Body density was estimated using air displacement plethysmography usingthe BODPOD® (COSMED, Rome, Italy). Prior to each test, the BODPOD wascalibrated according to the manufacturer's instructions using atwo-point calibration. Prior to testing, athletes were instructed towear tight fitting compression shorts and a swimming cap, as well as toremove all metal, including jewelry and watches. Body mass was measuredto the nearest 0.01 kg using the system's calibrated scale. All athleteswere instructed to sit in the chamber, breath normally, and to minimizeany movement. A minimum of two trials were performed. If measurementswere not within 150 ml of each other, a third trial was conducted.Thoracic gas volume was estimated using the BODPOD software, which usesstandard prediction equations and has demonstrated no differencecompared to measured lung volumes (McCrory M A, Molé P A, Gomez T D,Dewey K G, and Bernauer E M. Body composition by air-displacementplethysmography by using predicted and measured thoracic gas volumes.Journal of Applied Physiology 84: 1475-1479, 1998).

Bioelectrical Impedance Analysis

Total body water (TBW) was determined using multi-frequencybioelectrical impedance analysis (BIA) using the InBody® 570 BodyComposition Analyzer device (Biospace, Inc., Seoul, Korea). Bodycomposition from BIA is obtained from the measures of resistance andreactance when an electrical current travels throughout the body. Priorto each assessment the participants' hands and feet were thoroughlycleaned with InBody® provided tissues. Age, height, and sex weremanually entered, while a scale positioned within the device assessedbody mass. The participant was then instructed from the software tostand fully erect on the measurement electrodes situated on the platformand to hold hand electrodes, with arms extended, without touching thesides of their body. Participants were asked to refrain from moving ortalking until the assessment was completed. It has previously been shownthat BIA is a valid measurement tool for determining TBW when comparedto a deuterium oxide technique (Anderson L J, Erceg D N, and Schroeder ET. Utility of multi-frequency bioelectrical impedance compared todeuterium dilution for assessment of total body water. Nutrition &dietetics 72: 183-189, 2015).

Three-Compartment Model (3C-W)

The criterion percent body fat (% BF) was estimated using the threecompartment-water (3C-W) model described by Siri (Siri W E. The grosscomposition of the body. Adv Biol Med Phys 4: 513, 1956). The equationincludes measurements of body density (from the BODPOD), TBW (from theBIA), and body mass (BM). The equation for % BF is listed below:% BF=[(2.118/Body density)−(0.78×TBW(L)/BM(kg))−1.354]×100  Equation (1)

Muscle Ultrasonography

Non-invasive measurements of muscle thickness (MT) were collected usingB-mode ultrasound imaging with a 12 MHz linear probe (General ElectricLOGIQ P5, Wauwatosa, Wis.). Measurements for the rectus femoris (RF)were taken at 50% of the distance from the anterior, inferiorsuprailliac spine to the most proximal point of the patella (Jajtner AR, Hoffman J R, Scanlon T C, Wells A J, Townsend J R, Beyer K S, MangineG T, McCormack W P, Bohner J D, and Fragala M S. Performance and musclearchitecture comparisons between starters and nonstarters in NationalCollegiate Athletic Association Division I women's soccer. The Journalof Strength & Conditioning Research 27: 2355-2365, 2013). Vastuslateralis (VL) measurements were taken in the same fashion as previouslystated; however, the sampling location is determined by 50% thestraight-line distance between the greater trochanter and the lateralepicondyle of the femur (Abe T, Fukashiro S, Harada Y, and Kawamoto K.Relationship between sprint performance and muscle fascicle length infemale sprinters. Journal of physiological anthropology and appliedhuman science 20: 141-147, 2001). Prior to image collection,participants laid supine for 5 minutes and the probe was coated with awater-based conduction gel (Arroyo E, Stout J R, Beyer K S, Church D D,Varanoske A N, Fukuda D H, and Hoffman J R. Effects of supine restduration on ultrasound measures of the vastus lateralis. Clinicalphysiology and functional imaging 38: 155-157, 2018). For measurementsof MT, the probe was oriented longitudinally in the sagittal planeparallel to the muscle tissue without depressing the skin. Once imageswere collected, analysis was completed using Image J software (version1.45s; National Institutes of Health, Bethesda, Md., USA). MT wasdetermined from the still image as the distance between the inferiorborder of the superficial aponeurosis and the superior border of thedeep aponeurosis. Intraclass correlation coefficients (ICC_(3,k)) andstandard error of measurements (SEM) for the ultrasound technician werecalculated for the RF MT (ICC_(3,k)=0.99, SEM_(3,k)=0.07, MD=0.19 cm)and VL MT (ICC_(3,k)=0.99, SEM_(3,k)=0.01, MD=0.03 cm) from analysis of10 individuals separated by 24 hours.

Dynamic Strength Testing

One-repetition maximum (1RM) strength was assessed in squat and deadlift exercises. All 1RM testing was performed using methods previouslydescribed (Hoffman, J. Norms for Fitness, Performance, and Health (HumanKinetics: Champaign, Ill., 2006.) Prior to testing, each athletecompleted a general warm-up led by the strength and conditioning coach,which included jogging and a dynamic warm-up. Each athlete performed twowarm-up sets using a resistance of approximately 40-60% and 60-80% ofher perceived maximum, respectively. For each exercise 3-4 subsequenttrials were performed to determine the 1-RM. A 3-5 min rest period wasprovided between each trial. Trials not meeting the range of motioncriteria for each exercise or where proper technique was compromisedwere discarded.

Performance Testing

Ten-Yard Sprint

The athletes then completed a standardized general and dynamic warm-upthat was consistent with their normal training habits and led by eachteams' strength and conditioning coach. A pair of cones and tape affixedto the floor were positioned to denote the “starting line”. The athleteswere instructed to take their preferred starting stance at the startingline and to begin each maximal trial at their ready. The best of threetrials was recorded and used for analysis.

Pro-Agility Test

For the pro-agility test, three cones were placed parallel, five metersapart. The athletes set up for the test in a straddle position facingthe middle cone. On their ready, the athletes were instructed to pivotto their right and accelerate as quickly as possible to a cone 5 m awayand then upon touching the first cone, pivot again to their left andsprint the 10 m distance to the furthest cone. Upon touching this cone,the athletes once again pivoted to the right to return to the middlecone as quickly as possible. During each change in direction, theathletes were asked to touch the ground next to the cone. Trials wherethe athlete failed to touch the ground were discarded. Athletes wereallowed three attempts and the fastest time measured in seconds wasrecorded.

Standing Long Jump

Standing long jump performance was assessed using a pre-marked (±0.5 in)commercial mat (Sportime, LLC, Norcross, Ga., USA). Prior to the test,each athlete stood with both feet placed in the marked “starting area”on the mat. Athletes were instructed to perform a maximal horizontallong jump. Standing long jump distance was determined by furthestdistance reached following 3 maximal countermovement jump attemptsperformed from a standing position with feet shoulder width apart.

Results

Changes in strength, performance and body composition are presented inTable 2. There were no group differences observed between PRO and PL forany measure of strength, performance or body composition. Collectively,significant improvements (p<0.001) were observed in squat 1RM, deadlift1RM, pro-agility, and standing long jump as a result of 12-weeks ofoffseason training while no improvement (p=0.312) in 10-yard sprint timewas found. Additionally, both groups experienced significantly increased(p<0.001) RF and VL muscle thickness following training while noimprovements were seen following Body Fat % (p=0.332).

While no differences in training outcomes were observed, probioticsupplementation still may foster a more favorable physiological statefor recovery and adaptation.

In the current study, we observed no differences in any measure ofphysical performance between groups. Additionally, we found nopreferential effects of probiotic supplementation on muscle thicknessand body composition.

TABLE 2 Strength, Performance, and Body Composition Changes Following12- weeks of Offseason Training 95% Confidence Interval Variable PRECovariate POST F p η² Lower Upper Squat 1RM (kg) PRO 116.8 ± 17.1  124.9141.8 ± 11.2  .459 .505 .020 139.2 159.4 PL 133.0 ± 32.0  162.2 ± 40.0 143.6 164.7 Deadlift 1RM (kg) PRO 139.9 ± 12.2  151.3 169.4 ± 21.0  .375.547 .019 172.2 188.9 PL 162.8 ± 40.5  188.0 ± 39.1  168.7 185.2Standing Long PRO 2.46 ± 0.17 2.50 2.55 ± 0.21 .046 .833 .003 2.53 2.64Jump (m) PL 2.54 ± 0.28 2.64 ± 0.19 2.54 2.66 Pro-Agility (sec) PRO 4.62± 0.17 4.60 4.49 ± 0.22 1.152 .300 .071 4.41 4.55 PL 4.58 ± 0.20 4.50 ±0.23 4.46 4.60 10yd Sprint (sec) PRO 1.99 ± 0.86 1.86 1.69 ± 0.12 .852.371 .054 1.63 1.77 PL 1.70 ± 0.11 1.66 ± 0.09 1.57 1.73 Body Fat (%)PRO 14.7 ± 5.6  14.3 14.9 ± 4.8  2.119 .161 .096 13.7 15.7 PL 14.0 ±4.9  13.4 ± 4.8  12.9 14.6 RF Muscle PRO 2.39 ± 0.44 2.44 2.51 ± 0.47.166 .687 .008 2.49 2.64 Thickness (cm) PL 2.50 ± 0.28 2.60 ± 0.29 2.462.62 VL Muscle PRO 1.73 ± 0.23 1.79 1.78 ± 0.23 .513 .481 .023 1.81 1.89Thickness (cm) PL 1.86 ± 0.33 1.93 ± 0.33 1.83 1.91 Data presented asmean ± SD.

Example 3. Biomarker Level in Saliva and in Blood

Saliva Sampling

Saliva and blood samples were obtained at two time points throughout thestudy (PRE, POST). All biochemical samples at POST were taken at thesame time of day as PRE to avoid potential confounding influence ofdiurnal variations. Prior to saliva sampling, all athletes rested in aseated position for 5 minutes. With an initial swallow to empty themouth, unstimulated whole saliva was collected by expectoration into apre-weighed vial for with eyes open, head tilted slightly forward andmaking minimal orofacial movement. Study personnel then documented thesaliva collection duration and weight of the sample. Saliva flow rate(mL/min) was determined by weighing with saliva density assumed to be1.0 g/mL (Chicharro J L, Lucía A, Pérez M, Vaquero A F, and Ureña R.Saliva composition and exercise. Sports medicine 26: 17-27, 1998). Aftercollection, the sample tube was centrifuged at 3000 g for 15 min toremove cellular debris and which can negatively impact the accuracy ofanalysis (Schipper R G, Silletti E, and Vingerhoeds M H. Saliva asresearch material: biochemical, physicochemical and practical aspects.Archives of oral biology 52: 1114-1135, 2007). The supernatant was thenaliquoted and stored frozen at −80° C. for later analysis.

Biochemical Analyses of Saliva Samples

Duplicate saliva samples were analyzed for secretory IgA and IgMconcentrations using enzyme-linked immunosorbent assay (ELISA) kits(IgA: Salimetrics, State College, Pa., USA; IgM: Abcam, Toronto,Ontario, Canada). The intra-assay coefficient of variation for salivaIgA was 3.31% and 7.54% for IgM. The IgA and IgM secretion rate was thencalculated by multiplying the concentration by the saliva flow rate.

Blood Sampling

These blood samples were obtained using a single-use disposable needlewith the athlete in a supine position for at least 15 minutes beforesampling. All blood samples were collected into two Vacutainer® tubes,one containing no anticlotting agent (6 mL) and the second containingK2EDTA (6 mL). The blood in the first tube was centrifuged immediatelyat 3000 g for 15 min while the second tube was allowed to clot at roomtemperature for 30 min and subsequently centrifuged at 3000 g for 15min. The resulting plasma and serum were placed into separately labeledmicrocentrifuge tubes and frozen at −80° C. for later analysis.

Biochemical Analyses of Blood Samples

Circulating plasma concentrations of TNF-α and serum concentrations ofIL-10, zonulin, testosterone, and cortisol were assayed via commerciallyavailable ELISA kits (ALPCO, Salem, N.H., USA). To limit interassayvariability, all samples for a particular assay were thawed once, andanalyzed by the same technician using a FLUOstar Omega spectrophotometer(BMGLabtech, Ortenberg, Germany). All samples were analyzed in duplicatewith a mean coefficient of variation of 4.05% for TNF-α, 7.45% forIL-10, 4·10% for zonulin, 4.89% for testosterone, and 3.48% forcortisol.

Results

Changes in biochemical markers are presented in Table 3.

TABLE 3 Changes in Biochemical Markers Following 12-weeks of OffseasonTraining 95% Confidence Interval Variable PRE Covariate POST F p η²Lower Upper TNF-α PRO 2.32 ± 0.93 2.37 2.07 ± 0.76 5.857 .024* .210 1.692.49 (pg/mL) PL 2.42 ± 1.49 2.78 ± 0.95 2.35 3.18 LN IL-10 PRO 2.79 ±0.97 2.95 2.89 ± 1.08 .032 .860 .001 2.89 3.22 (pg/mL) PL 3.12 ± 0.883.27 ± 1.02 2.91 3.25 Zonulin PRO 10.59 ± 2.11  10.14 10.78 ± 2.23  .010.921 <0.001 9.68 11.04 (ng/mL) PL 9.67 ± 4.32 9.86 ± 4.27 9.60 11.02Testosterone PRO 15.3 ± 6.59 15.7 15.8 ± 6.50 1.89 .183 0.79 14.8 17.4(nmol/L) PL 16.2 ± 4.56 17.8 ± 4.46 16.0 18.8 Cortisol PRO 656.3 ± 237.7662.8 579.4 ± 183.2 3.411 .078 .134 488.9 678.0 (nmol/L) PL 669.9 ±224.1 709.5 ± 247.4 606.6 803.5 T/C Ratio PRO .024 ± .009 .025 .030 ±.013 .464 .503 .021 .024 .036 PL .025 ± .008 .027 ± .009 .020 .033 TotalWBC PRO 5.97 ± 1.50 5.84 7.08 ± 1.85 .235 .632 .011 5.95 8.21 (×10⁹/L)PL 5.71 ± 1.31 7.46 ± 2.00 6.28 8.64 SIgA Secreation PRO 105.2 ± 56.4 123.1 176.6 ± 86.5  1.585 .222 .070 138.6 236.7 Rate (μg/min) PL 141.1 ±97.2  156.1 ± 98.3  96.0 194.1 LN SIgM PRO 8.11 ± 1.45 8.07 8.84 ± 1.07.452 .509 .021 8.32 9.30 Secreation Rate PL 8.02 ± 1.40 8.55 ± 1.50 8.109.07 (μg/min) Data presented as mean ± SD. LN = natural logtransformation. *significantly different from PL

TNF-α concentrations were significantly (F=5.859, p=0.024 η²=0.020)lower in PRO (Δ: −0.25±1·10 pg/mL, p=0.453) compared to PL (Δ: +0.36pg/mL, p=0.160).

The results obtained from this this embodiment indicate that 12-weeks ofprobiotic supplementation attenuated increases in TNF-α which wereobserved in the placebo group.

There were no other significant group differences in any otherbiochemical markers examined. However, a trend (F=3.41, p=0.078,η²=0.134) for lower cortisol concentrations in PRO (Δ: −76.9±222.1nmol/L, p=0.235) compared to PL (Δ: +39.6±126.03 nmol/L, p=0.300) wasobserved at POST. Collectively, significant increases were observed fortestosterone (p=0.045), LN IL-10 (p=0.048), SIgA rate (p=0.031), and LNSIgM rate (p=0.002) following 12-weeks of offseason training acrossgroups. No major effects over time were observed in any otherbiochemical marker.

While IL-10 concentrations in this embodiment did not differ betweengroups, significant elevations were seen as a result of the offseasontraining program.

No significant differences in testosterone (T), cortisol (C), or T:Cratio were observed between groups in this embodiment. Nevertheless, atrend was observed for decreased cortisol concentrations in theprobiotic group.

Furthermore, coupled with the probiotic attenuation of TNF-α in ourstudy, coinciding lower average cortisol levels in the probiotic groupindicate a better homeostatic balance for health, recovery, andphysiological adaptations.

In this embodiment, no significant differences were observed in plasmazonulin concentrations following our 12-week intervention.

Nevertheless, since no adverse effects of probiotic supplementation wereobserved, the findings of this embodiment provide additional support forthe possible benefits of probiotic supplementation in an athleticpopulation. Overall, during a time period where multiple stressors werepresent, probiotic supplementation may alter cytokine production, e,g.TNF-α, in male collegiate athletes.

Further, it is expected that probiotic supplementation using B.subtilis, containing supplements as described herein will providephysical and/or physiological benefits in a human male population inconjunction with reduced TNF-α levels in serum.

In certain embodiments, the compositions comprising Bacillus subtiliscan include one or more dry carriers selected from the group consistingof trehalose, maltodextrin, rice flour, microcrystalline cellulose,magnesium stearate, inositol, fructooligosaccharide,galactooligosaccharide, dextrose, and the like. In certain embodiments,the dry carrier can be added to the compositions comprising Bacillussubtilis in a weight percentage of from about 1% to about 95% by weightof the composition.

In certain embodiments, the compositions comprising Bacillus subtiliscan include one or more liquid or gel-based carriers, selected from thegroup consisting of water and physiological salt solutions, urea,alcohols and derivatives thereof (e.g., methanol, ethanol, propanol,butanol), glycols (e.g., ethylene glycol, propylene glycol), and thelike; natural or synthetic flavorings and food-quality coloring agents,all compatible with the organism; thickening agents selected from thegroup consisting of corn starch, guar gum, xanthan gum, and the like;one or more spore germination inhibitors selected from the groupconsisting of hyper-saline carriers, methylparaben, guargum,polysorbate, preservatives, and the like. In certain embodiments, theone or more liquid or gel-based carrier(s) can be added to thecompositions comprising Bacillus subtilis in a weight/volume percentageof from about 0.6% to about 95% weight/volume of the composition. Incertain embodiments, the natural or synthetic flavoring(s) can be addedto the compositions comprising Bacillus subtilis in a weight/volumepercentage of from about 3.0% to about 10.0% weight/volume of thecomposition. In certain embodiments, the coloring agent(s) can be addedto the compositions comprising Bacillus subtilis in a weight/volumepercentage of from about 1.0% to about 10.0% weight/volume of thecomposition. In certain embodiments, the thickening agent(s) can beadded to the compositions comprising Bacillus subtilis in aweight/volume percentage of about 2% weight/volume of the composition.In certain embodiments, the one or more spore germination inhibitors canbe added to the compositions comprising Bacillus subtilis in aweight/volume percentage of about 1% weight/volume of the composition.

Delivery System

Suitable dosage forms include tablets, capsules, solutions, suspensions,powders, gums, and confectionaries. Sublingual delivery systems include,but are not limited to, dissolvable tabs under and on the tongue, liquiddrops, and beverages. Edible films, hydrophilic polymers, oraldissolvable films, or oral dissolvable strips can be used. Other usefuldelivery systems comprise oral or nasal sprays or inhalers, and thelike. Suitable dosage forms include tablets, capsules, solutions,suspensions, powders, gums, and confectionaries. Sublingual deliverysystems include, but are not limited to, dissolvable tabs under and onthe tongue, liquid drops, and beverages. Edible films, hydrophilicpolymers, oral dissolvable films, or oral dissolvable strips can beused. Other useful delivery systems comprise oral or nasal sprays orinhalers, and the like.

For oral administration, probiotics may be further combined with one ormore solid inactive ingredients for the preparation of tablets,capsules, pills, powders, granules, or other suitable dosage forms. Forexample, the active agent may be combined with at least one excipientselected from the group consisting of fillers, binders, humectants,distintegrating agents, solution retarders, absorption accelerators,wetting agents, absorbents, and lubricating agents. Other usefulexcipients include, but are not limited to, magnesium stearate, calciumstearate, mannitol, xylitol, sweeteners, starch, carboxymethylcellulose,microcrystalline cellulose, silica, gelatin, silicon dioxide, and thelike.

In certain embodiments, the components of compositions administeredaccording to the methods of the present disclosure, together with one ormore conventional adjuvants, carriers, or diluents, may thus be placedinto the form of pharmaceutical compositions and unit dosages thereof.Such forms include: solids, and in particular, tablets, filled capsules,powder and pellet forms; liquids, and in particular, aqueous ornon-aqueous solutions, suspensions, emulsions, elixirs; and capsulesfilled with the same; all for oral use, suppositories for rectaladministration, and sterile injectable solutions for parenteral use.Such pharmaceutical compositions and unit dosage forms thereof maycomprise conventional ingredients in conventional proportions, with orwithout additional active compounds or principles, and such unit dosageforms may contain any suitable effective amount of the active ingredientcommensurate with the intended daily dosage range to be employed.

The components of the compositions administered according to the methodsof the present disclosure can be administered in a wide variety of oraland parenteral dosage forms. It will be obvious to those skilled in theart that the following dosage forms may comprise, in certainembodiments, as the active component, either a chemical compound of thepresent disclosure or a pharmaceutically acceptable salt of a chemicalcompound of the present disclosure.

For preparing pharmaceutical compositions to be administered accordingto the methods of the present disclosure, pharmaceutically acceptablecarriers can be either solid or liquid. Solid form preparations includepowders, tablets, pills, capsules, cachets, suppositories, anddispersible granules. A solid carrier can be one or more substances thatmay also act as diluents, flavoring agents, solubilizers, lubricants,suspending agents, binders, preservatives, tablet disintegrating agents,or encapsulating materials.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingcapacity in suitable proportions and compacted in the shape and sizedesired.

In certain embodiments, powders and tablets administered according tomethods of the present disclosure preferably may contain from five orten to about seventy percent of the active compound. Suitable carriersare magnesium carbonate, magnesium stearate, talc, sugar, lactose,pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as carrier providing acapsule in which the active component, with or without additionalcarriers, is surrounded by a carrier, which is thus in association withit. Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid formssuitable for oral administration.

Liquid preparations include, but are not limited to, solutions,suspensions, and emulsions, for example, water or water-propylene glycolsolutions. For example, parenteral injection liquid preparations can beformulated as solutions in aqueous polyethylene glycol solution. Incertain embodiments, chemical compounds administered according tomethods of the present disclosure may thus be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose foradministration in ampoules, pre-filled syringes, small-volume infusion,or in multi-dose containers with an added preservative. The compositionsmay take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and may contain formulation agents such as suspending,stabilizing, and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizing and thickening agents, as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,or other well-known suspending agents.

Compositions suitable for topical administration in the mouth include,but are not limited to: lozenges comprising the active agent in aflavored base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatin andglycerine or sucrose and acacia; and mouthwashes comprising the activeingredient in suitable liquid carrier.

Solutions or suspensions are applied directly to the nasal cavity byconventional means, for example, with a dropper, pipette, or spray. Thecompositions may be provided in single or multi-dose form. Incompositions intended for administration to the respiratory tract,including intranasal compositions, the compound will generally have asmall particle size, for example, of the order of 5 microns or less.Such a particle size may be obtained by means known in the art, forexample, by micronization.

The pharmaceutical preparations are preferably in unit dosage forms. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packaged tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself; or it can be the appropriate number of any of thesein packaged form.

Tablets, capsules, and lozenges for oral administration and liquids fororal use are preferred compositions. Solutions or suspensions forapplication to the nasal cavity or to the respiratory tract arepreferred compositions. Transdermal patches for topical administrationto the epidermis are preferred.

Further details on techniques for formulation and administration may befound in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (MackPublishing Co., Easton, Pa.).

Routes of Administration

The compounds may be administered by any route, including, but notlimited to, oral, sublingual, buccal, ocular, pulmonary, rectal, andparenteral administration, or as an oral or nasal spray (e.g.,inhalation of nebulized vapors, droplets, or solid particles).Parenteral administration includes, for example, intravenous,intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal,intravesical (e.g., to the bladder), intradermal, transdermal, topical,or subcutaneous administration. Also contemplated within the scope ofthe invention is the instillation of a pharmaceutical composition in thebody of the patient in a controlled formulation, with systemic or localrelease of the drug to occur at a later time. For example, the drug maybe localized in a depot for controlled release to the circulation, orfor release to a local site.

Pharmaceutical compositions of the invention may be those suitable fororal, rectal, bronchial, nasal, pulmonal, topical (including buccal andsub-lingual), transdermal, vaginal or parenteral (including cutaneous,subcutaneous, intramuscular, intraperitoneal, intravenous,intraarterial, intracerebral, intraocular injection, or influsion)administration, or those in a form suitable for administration byinhalation or insufflation, including powders and liquid aerosoladministration, or by sustained release systems. Suitable examples ofsustained release systems include semipermeable matrices of solidhydrophobic polymers containing the compound of the invention, whichmatrices may be in the form of shaped articles, e.g., films ormicrocapsules.

The use of the terms “a,” “an,” “the,” and similar referents in thecontext of describing the present invention (especially in the contextof the claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. Use of the term “about” is intended todescribe values either above or below the stated value in a range ofapproximately ±10%; in other embodiments, the values may range in valueabove or below the stated value in a range of approximately ±5%; inother embodiments, the values may range in value above or below thestated value in a range of approximately ±2%; in other embodiments, thevalues may range in value above or below the stated value in a range ofapproximately ±1%. The preceding ranges are intended to be made clear bycontext, and no further limitation is implied. All methods describedherein can be performed in any suitable order unless otherwise indicatedhere in or otherwise clearly contradicted by context. The use of any andall examples, or exemplary language (e.g., “such as”) provided herein,is intended merely to better illuminate the invention and does not posea limitation on the scope of the invention unless otherwise stated. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

While in the foregoing specification this invention has been describedin relation to certain embodiments thereof, and many details have beenput forth for the purpose of illustration, it will be apparent to thoseskilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention.

All references cited herein are incorporated by reference in theirentireties. The present invention may be embodied in other specificforms without departing from the spirit or essential attributes thereof,and, accordingly, reference should be made to the appended claims,rather than to the foregoing specification, as indicating the scope ofthe invention.

We claim:
 1. A method of reducing tumor necrosis factor-alpha (TNF-α) inhuman serum, comprising the steps of: (a) administering orally to ahuman a composition comprising Bacillus subtilis in a dose of from about1·10⁸ colony forming units (CFU) per day to about 1·10¹¹ CFU per day forabout 12 weeks; and (b) submitting the human to a resistance trainingprogram throughout the 12 weeks.
 2. The method of claim 1, wherein theresistance program includes workouts 2-3 times per week.
 3. The methodof claim 1, further comprising the steps of measuring TNF-α in bloodplasma of the human both before and after steps (a) and (b).
 4. Themethod of claim 1, wherein the composition comprises Bacillus subtilisin a dose of from about 1·10⁹ CFU per day to about 1·10¹⁰ CFU per day.5. The method of claim 4, wherein the TNF-α level is reduced by about0.2 pg/mL in serum.
 6. A method for treating or reducing inflammation ina human, comprising the steps of: (a) administering orally to the humanin need of such treatment a composition comprising Bacillus subtilis ina dose of from about 1·10⁸ CFU per day to about 1·10¹¹ CFU per day forabout 12 weeks, and (b) measuring TNF-α in blood plasma of the human,wherein the TNF-α level is reduced in serum.
 7. The method of claim 6,further comprising the step of: (b) submitting the human to a resistancetraining program throughout the 12 weeks.
 8. The method of claim 6,wherein the composition comprises Bacillus subtilis in a dose of fromabout 1·10⁹ CFU per day to about 1·10¹⁰ CFU per day.
 9. The method ofclaim 7, wherein the composition comprises Bacillus subtilis in a doseof from about 1·10⁹ CFU per day to about 1·10¹⁰ CFU per day.
 10. Themethod of claim 9, wherein the resistance program includes workouts 2-3times per week.
 11. The method of claim 10, further comprising the stepsof measuring TNF-α in blood plasma of the human both before and aftersteps (a) and (b).
 12. The method of claim 11, wherein the TNF-α levelis reduced by about 0.2 pg/mL in serum.